Quan Sun

Microsporidia are a group of intracellular and unicellular eukaryotic parasites, which can nearly infect all animals, including human beings. As the first identified microsporidia, Nosema bombycis is a world-wide threat for silkworm eggs production, it can cause the congenital infection via transovarial transmission. It is important for pathogenesis elucidation to unravel the molecular characteristics of N. bombycis proliferation and host immune responses to the congenital infection in embryo and larva stage. Here, we adopted dual RNA-seq approach to investigate and compare the dynamic molecular pattern of pathogen proliferation and host immune responses between diapause and non-diapause silkworm eggs. Our results showed the N. bombycis proliferation in non-diapause silkworm eggs is a continuous process, many parasites enter the sporogony stage at 2 days post-oviposition (dpo). For newly hatched larva (1dph), the abundance of pathogen mRNA sequences is up to 2.32% in non-diapause strain, far higher than 0.34% of diapause strain, the main reason is the hot HCl bath treatment at 24 hours post-oviposition for diapause silkworm eggs with the aim to free the egg diapause. As to immune responses, whatever for diapause strain or non-diapause strain, there is stronger immune responses to congenital infection in larva stage than that of embryo stage, however, the host immune responses to congenital infection are fairly different between non-diapause and diapause strains of silkworms, especially in embryo stage. We found the surprising “First day Chaos” that there are 6,071 differential expressed genes (DEGs) at 1 dpo for non-diapause strain between infection group and uninfected group, but decreases dramatically to 109 DEGs at 2 dpo. In non-diapause strain, the earliest DEGs of antimicrobial peptides were up-regulated at 1 dpo, then is 5 dpo with up-regulated lebocin, 7 dpo with morLP-B1, morLP-B4. For non-diapause strain, the well-established immune responses were observed in newly hatched larvae. On the contrast, for diapause strain, the earliest DEGs of AMPs appear at 5 dph, the mature immune responses are well established at 5 dph too. In non-diapause silkworms, we observed obvious pathogen’s regulation in the main immune pathways including Toll, IMD, JAK-STAT and melanization at the different steps such as immune recognition, signal modulation and transduction, effectors. Taken together, our results for the first time provide a global molecular view of microsporidia proliferation and innate immunity responses in a congenital infection system and provide some new insights into immune development and establishment in the embryo and early larva stage of Bombyx mori.

Microsporidia are obligate intracellular eukaryotic parasites that have significantly reduced genomes and that have lost most of their introns. In the current study, we characterized a gene in microsporidia Nosema bombycis, annotated as TRAPα (HNbTRAPα). The homologous of TRAPα are a functional component of ER translocon and facilitates the initiation of protein translocation in a substrate-specific manner, which is conserved in animals but absent from most fungi. The coding sequence of HNbTRAPα consists of 2226 nucleotides, longer than the majority of homologs in microsporidia. A 3′ RACE analysis indicated that there were two mRNA isoforms resulting from non-canonical alternative polyadenylation (APA), and the polyadenylate tail was synthesized after the C⁹⁵¹ or C¹¹⁶⁷ nucleotide, respectively. Indirect immunofluorescence analysis showed two different localization characteristics of HNbTRAPα, which are mainly located around the nuclear throughout the proliferation stage and co-localized with the nuclear in mature spores. This study demonstrated that the post-transcriptional regulation mechanism exists in Microsporidia and expands the mRNA isoform repertoire.

Microsporidia are a big group of single-celled obligate intracellular organisms infecting most animals and some protozoans. These minimalist eukaryotes lack numerous genes in metabolism and vesicle trafficking. Here, we demonstrated that the spore wall protein NbSWP12 of microsporidium Nosema bombycis belongs to Bin/Amphiphysin/Rvs (BAR) protein family and can specifically bind with phosphatidylinositol 3-phosphate [Ptdlns(3)P]. Since Ptdlns(3)P is involved in endosomal vesicle biogenesis and trafficking, we heterologous expressed NbSWP12 in yeast Saccharomyces cerevisiae and proved that NbSWP12 can target the cell membrane and endocytic vesicles. Nbswp12 transformed into Gvp36 (a BAR protein of S. cerevisiae) deletion mutant rescued the defect phenotype of vesicular traffic. This study identified a BAR protein function in vesicle genesis and sorting and provided clues for further understanding of how microsporidia internalize nutrients and metabolites during proliferation.